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Effects of doe–litter separation on endocrinological and
productivity variables in lactating rabbits
a ,
*
a b b a˜
E. Ubilla
, P.G. Rebollar , D. Pazo , A. Esquifino , J.M.R. Alvarino
a
´ ´ ´
Departamento de Produccion Animal, E.T.S.I. Agronomos, Universidad Politecnica, Ciudad Universitaria s /n, 28040 Madrid, Spain
b
´
Departamento de Bioquımica, Facultad de Medicina, Universidad Complutense, Ciudad Universitaria s /n, 28040 Madrid, Spain
Received 9 August 1999; received in revised form 2 February 2000; accepted 7 March 2000
Abstract
The objective of this study was to explore the effects of a doe–litter separation for 48 h before artificial insemination (AI) on various endocrinological and productivity variables determined in 24 doe rabbits. The control group (C) (n512) had free access to nursing and the separated group (S) (n512) were kept apart form their litters for 48 h prior to AI on day 11 of the lactation period. Lower plasma prolactin (PRL) concentrations were observed 24 h after the doe–litter separation (P,0.04). The S does had increased plasma oestradiol (E ) concentrations 48 h after the doe–litter separation, compared with2 preseparation values of the same group and with C does (P,0.0001). A higher proportion of does showing high sexual receptivity was observed among the S group compared with the C group (P,0.05). The suckling episode ranged from 3 to 5 min in S does. Teat stimulation by suckling caused an immediate increase in PRL in S does (P,0.0001). PRL remained high until 7 h after suckling in S does, compared with C does (P,0.04). The doe–litter separation resulted in a mean (6S.E.M.) reduction in litter weight of 217 (622.1) g (P,0.02). No kits died in either group during the separation period. The litter weight on day 21 after parturition and milk production / doe were not affected by treatment. When analysing the subsequent litters, no differences were observed between S and C does in kindling rate, litter size at birth, number of dead kits / litter, litter size at weaning and number of kits dead from birth to weaning. The results suggest that a transient doe–litter separation causes an increase in the proportion of does showing signs of oestrus and may have a major influence on fertility. These effects may be induced by the absence of suckling episodes together with the higher plasma E and lower PRL2 concentrations seen in the S does prior to AI. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Doe–litter; Prolactin; Oestradiol; Sexual receptivity; Productivity; Rabbits
1. Introduction anoestrous periods, constituting a disadvantage when attempting to establish synchronised reproduction The reproductive performance of lactating doe and the use of artificial insemination (AI). Kindling rabbits is characterised by irregular oestrous and rates (KR) obtained in artificially inseminated lactat-ing doe rabbits appear to be affected by frequent variations in sexual receptivity (SR) during the *Corresponding author. Tel.: 134-1-549-1880; fax: 1
34-1-lactation period (Castellini et al., 1998). Receptive 549-9763.
E-mail address: eubilla@pan.etsia.upm.es (E. Ubilla). does are reported to have high plasma oestradiol17-b
(E ) concentrations (Ubilla and Rebollar, 1995);2 breed doe rabbits. Animals were housed in research these show higher KR and litter size at birth than facilities of the Animal Production Department. They
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non-receptive does (Theau-Clement and Roustan, were maintained under controlled light–dark cycles 1992). The reduction in fertility and SR rates ob- (16:8, L:D), housed in individual metal cages, fed ad served during the lactation period appeared to be libitum using a commercial pelleted diet (Lab Rabbit
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influenced by hormonal antagonisms between blood Chow, Purina Mills, Torrejon de Ardoz-Madrid, prolactin (PRL) and gonadotrophin concentrations Spain) and had free access to tap water. On day 1 (Fortun and Bolet, 1995; Ubilla and Rebollar, 1995). after parturition, litter size was standardised to 8–9 Recently, several studies on stimulating ovarian by adding or removing kits to assure similar lactation activity in lactating doe rabbits have demonstrated conditions during the experiment.
the influence of the separation of does from their
litters for short periods of time prior to AI (Pavois et 2.2. Experimental design ˜
al., 1994; Alvarino et al., 1998; Maertens, 1998).
These methods, especially after a doe–litter sepa- The lactating does were randomly allocated to one ration of at least 36 h before AI, improved KR and of two groups of 12 animals: (1) a control (C) group litter size at birth, with results similar to those with free access to nursing and (2) a separated (S) obtained when administering hormonal treatments, group in which the does were separated from their such as prostaglandin F2a(PGF2a) or pregnant mare litters by means of a metal screen for 48 h prior to serum gonadotrophin (PMSG), before AI (Facchin et AI (Luzi and Crimella, 1998), i.e. from day 9 until al., 1992; Pavois et al., 1994). Litter size at birth day 11 of the lactation period.
appeared to be reduced when AI was performed on Both groups were inseminated on day 11 of the day 4 after delivery in normal lactating does, com- lactation period using 1 ml / doe of the same fresh pared with does separated from their litters for 48 h semen pooled from several males and diluted using a
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before AI (Alvarino et al., 1998). These results commercial extender (MA 24, Ovejero, Leon, 6 indicate an antagonism between reproduction and Spain). Each dose contained at least 25310 sper-lactation during the first few days of the sper-lactation matozoa / ml. Ovulation was induced by gonadorelin,
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period (Alvarino et al., 1998). a synthetic analogue of gonadotrophin-releasing hor-˜ The above references suggest that a transient doe– mone (GnRH; 20 mg / doe, i.m.; Ovejero) (Alvarino litter separation in lactating doe rabbits may induce et al., 1998). Separated does received AI immedi-several endocrinological changes that could affect ately after nursing and blood sampling (about 10 min reproductive performance, probably by stimulating after the separation period ended). Control does the activity of the pituitary and ovary. Nevertheless, nursed their young at least 4 h before AI and the mechanisms involved in these events are not yet received AI after blood sampling. Pregnancies were
clearly understood. diagnosed by abdominal palpation on day 12 after
This study was therefore designed to investigate AI.
the changes occurring in plasma PRL and E con-2 In order to study the influence of separation on centrations after the separation of does from their plasma E2 and PRL concentrations, blood samples litters for a short period of time before AI, as well as were collected in both groups from the marginal ear the influence on SR, change in litter weight, and vein into heparinized tubes, and immediately cen-litter survival. Several other reproductive variables trifuged at 1000 g for 10 min at 88C. Plasma was obtained after parturition were also determined. stored at 2208C until analysed. The blood samples were collected at 24-h intervals from day 9 to 11 of the lactation period. To study the PRL secretion 2. Materials and methods pattern after suckling, sampling was also performed at the following times: 1, 1.5, 2, 2.5, 3, 3.5, 4 and 7 h
2.1. Animals after AI. In S does the first sample obtained on day
litters at least 4 h before sampling on day 9. All C The titre of antibody used was 1:62,500. The PRL rabbits nursed their litters at least 4 h before the standard used was RbPRL-RP-1. Hormone was
la-125
second and third blood samples were collected (on belled with I by the chloramine-T method (Green-days 10 and 11, respectively). No does of either wood et al., 1963). The volume of plasma for PRL group nursed their litters after AI until the last determinations was 10 ml. Staphylococcus aureus
´
sample was collected 7 h later. (prepared by the Autonoma University, Department Sexual receptivity was assessed based on the of Plant Physiology, U.A.M., Madrid, Spain) was turgidity and colour of the vulva (McNitt and used to precipitate the bound fraction (Ubilla et al., Moody, 1989; Ubilla and Rebollar, 1995) before AI. 1992). All samples were measured in the same assay The does were categorised into three levels: high SR run to avoid inter-assay variations. The sensitivity of (HSR), medium SR (MSR) and low SR (LSR) the assay for PRL was 0.125 ng / ml and the intra-(Rodriguez et al., 1989; Ubilla and Rebollar, 1995). assay coefficient of variation was ,5%. The intra-This study was performed according to the CEE assay coefficient was calculated using one pool of Council Directive 86 / 609 (1986) for care of ex- plasma measured ten times in the same assay. The perimental animals. mean (6S.E.M.) concentration was 106.964.1 ng / ml. Plasma E concentrations were determined using2
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2.3. Variables measured in the current litters a commercial I RIA kit (ICN Pharmaceuticals Diagnostics Division, Costa Mesa, CA, USA; Lot no. The influence of the transient doe–litter separation E2 K9917). Plasma E2 concentrations were ex-on litter survival was observed every 8 h (from 09:00 pressed as pg / ml. All samples were analysed in the h on day 9 to 09:00 h on day 11 of lactation). same assay. The sensitivity of the assay was 0.74 The change in litter weight after separation was pg / ml, and the intra-assay coefficient of variation evaluated by comparing the litter weights on day 9 was ,7%. The intra-assay coefficient was calculated versus day 11 of the same group. The duration of the using one pool of plasma measured 10 times in the suckling episode before AI on day 11 was measured same assay. The mean (6S.E.M.) concentration was in the S group at the end of the separation period. 35.862.5 pg / ml.
The litter weight was determined on day 21 after
parturition in both groups. Milk production / doe was 2.6. Statistics estimated for a lactation period of 30 days in both
groups by means of the following regression: y5 All analyses were conducted using the statistical 2
276.9111.831x; r 50.846 (P,0.001); ( y5milk analysis system (SAS, version 6.12; 1990). A non-production for a lactation period of 30 days; x5litter parametric procedure (PROC CATMOD) was used to weight on day 21 after parturition) (Torres et al., identify significant effects of treatment on the
vari-1979). ables that did not approximate to a normal
dis-tribution (PRL and E2 concentrations, SR, KR, 2.4. Variables measured in the subsequent litters number of dead kits / litter, litter size at weaning, number of kits dead from birth to weaning, and The KR, litter size at birth, number of dead kits / change in litter weight after the separation from the litter, litter size at weaning and number of kits dead does). Means were compared using the CONTRAST from birth to weaning was checked after parturition. procedure. Milk production / doe and litter size at birth variables were analysed by one-way analysis of 2.5. Hormone analyses variance followed by the Duncan multiple range test
(SAS, 1990). Plasma PRL concentrations were measured in
duplicate samples by a specific radioimmunoassay
(RIA) method (Ubilla et al., 1992) using AFP- 3. Results 991086 antibody supplied by the National Institute of
studied. Separated does showed lower plasma PRL mean reduction (P,0.02) in litter weight, while concentrations 24 h after doe–litter separation com- litter weight increased (P,0.02) from days 9 to 11 pared with preseparation values of the same group of the lactation period in C does.
and with the C does (P,0.04). Higher plasma PRL No kits died in either group during the separation concentrations were observed in S does after nursing period before AI. No differences were observed compared with the C does (P,0.0001). PRL con- between S and C does in litter weight on day 21 after centrations remained high from 1 to 7 h after parturition and milk production / doe (Table 1). The suckling compared with C does (P,0.001 to P, suckling episodes ranged from 3 to 5 min in S does.
0.04) (Fig. 1). No differences were observed between S and C does
Mean plasma E2 concentrations were similar in KR, litter size at birth, number of dead kits / litter, among C does during the period studied. The S does litter size at weaning and number of kits dead from showed increased plasma E2 concentrations 48 h birth to weaning in the subsequent litters (Table 1). after the doe–litter separation compared with the
preseparation values of the same group and with the
C does (P,0.0001) (Fig. 2). 4. Discussion
A higher proportion of HSR does was observed
among the S does compared with the C does (P, Plasma PRL concentrations measured in both
0.05) (Fig. 3). groups are consistent with those previously reported
The doe–litter separation before AI resulted in a during the lactation period (Fuchs et al., 1984; Ubilla
Fig. 2. Plasma oestradiol-17b (E ) concentrations in control (2 h) and separated (o) does. The arrow indicates the time of artificial insemination (AI). Each column represents the mean6S.E.M. of 12 samples. The separated does had higher E concentrations 48 h after the2
doe–litter separation compared with the preseparation values of the same group and with the control does (****, P,0.0001).
Fig. 3. Sexual receptivity (SR) in control and separated does. A higher proportion of high SR (HSR) does were observed in the separated group compared with the control group (*, P,0.05) (MSR5medium SR does; LSR5low SR does).
Table 1
Effect of doe–litter separation for 48 h before artificial insemination on mean (6S.E.M.) reproductive and productive variables in nursing doe rabbits
Separated does Control does
Current litter
Litter survival after separation (%) 100 100
a a
Change in litter weight after separation (g) 2217.6622.1 1302627.1 Litter weight on day 21 after parturition (g) 2402.16129.4 2234.36140.7
Milk production / doe (g) 46756150.4 43686135.1
Subsequent litter
Kindling rate (%) 100 83.3
Litter size at birth 9.0860.41 9.2060.39
Number of dead kits / litter 0.5160.13 0.4360.11
Number of kits dead from birth to weaning 1.1760.09 1.6760.11
Litter size at weaning (on day 30 after parturition) 7.460.19 7.160.24
a
Litter weights of the same group were compared between day 9 and 11 (P,0.02).
periods (Maertens, 1998). These effects could ex- rose to 10 times the previous mean values (detected plain the higher proportion of HRS does found in the on day 10 of lactation) in S does, confirming the S group. Similar findings were observed after a 40-h PRL release during suckling that occurs in response doe–litter separation or after PMSG treatments to physical stimulation of the nipples (McNeilly and
(Maertens, 1998). Friesen, 1978). This response was followed by
h after suckling. Similar PRL secretion patterns Although the litter weight was reduced by 15.5% during the early lactation period were previously after 48 h of doe–litter separation, all kits survived observed after a once daily suckling episode (Muc- and the effect of separation on litter weight was not cioli et al., 1982; Fuchs et al., 1984). The suckling detected on day 21 after parturition. Similarly, episode intervals observed in S does are in agree- Maertens (1998) found that the loss of weight of the ment with previous results (Grosvenor et al., 1967). litters was 7–8% after 40 h of separation. Several Compared with other animals, rabbit does normally authors have suggested that the loss in the litter nurse their litters once daily and the kits suckle weight observed could be partially compensated for
˜
during a 2–5 min uninterrupted suckling period during the fattening period (Alvarino et al., 1998). (Grosvenor et al., 1967; McNitt, 1992), whereas the Our study did not show any effect of the treatment rat nurses several times per day and the litters suckle on milk production / doe.
in an intermittent mode (Wakerley and Lincoln, The litter size at weaning and the number of kits 1971). In the rat, PRL secretion declines as soon as dead from birth to weaning was not negatively suckling stops (Grosvenor and Whitworth, 1974). affected by the treatment. These results are in Therefore, the differences in PRL secretion between agreement with those described by other authors these species could be a response to different suckl- (Castellini et al., 1998; Maertens, 1998).
ing patterns (Fuchs et al., 1984). The sustained PRL In summary, the results suggest that a doe–litter release observed in the rabbit may explain the separation for 48 h before AI causes an increase in essential lactogenic role that this hormone plays in the proportion of does showing HSR signs and may the maintenance of lactation (Deis et al., 1980; Fuchs have a major influence on the fertility of these does. et al., 1984). Frequent suckling episodes with short These effects are probably due to the absence of periods of elevated PRL release are necessary to start suckling episodes together with the higher plasma E2 and maintain lactation in other animals (Tolis et al., and lower PRL concentrations seen in the S does. 1974; Djiane et al., 1977) whereas, in doe rabbits, The suckling episodes ranged from 3 to 5 min and the sustained PRL release appears to be able to provoked a sustained PRL release of longer duration induce and maintain milk yield throughout lactation than that observed in normally lactating does. This (Fuchs et al., 1984). Other relevant aspects involved sustained PRL release may be involved with the with maternal behaviour may be induced by the maintenance of lactation and maternal behaviour. sustained PRL secretion, since does showing lower Although the litter weight decreased after the doe– plasma concentrations after parturition refuse to litter separation, all kits survived after separation and nurse their litters (McNeilly and Friesen, 1978). In no effect on the litter weight was detected on day 21 this study, the sustained PRL release observed in S after parturition. Milk production / doe was not af-does was detected until the last sampling, 7 h after fected by the treatment. The KR, litter size at birth, suckling. These results suggest a longer PRL release number of dead kits / litter, litter size at weaning and in does previously separated from their litters for a number of kits dead from birth to weaning, were not short period of time, than that found after suckling affected by the treatment in the subsequent litters. during a normal lactation (Fuchs et al., 1984; Ubilla Further information on the ovarian activity induced
et al., 1992). by a transient separation of does from their litters
The KR and litter size at birth were not affected appears to be necessary in order to confirm the by the doe–litter separation, and were consistent stimulatory actions of separation on SR and fertility.
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with previous results (Alvarino et al., 1998). Al-though no statistical differences for KR were
ob-tained, all S does became pregnant after AI com- Acknowledgements pared with 10 / 12 C does. This could be significant if
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